Preterm birth is a major public health burden that remains the leading cause of neonatal morbidity and mortality worldwide. Our long-term goal is to determine the mechanisms that disrupt the normal timing for parturition and lead to preterm birth. Numerous factors influence the likelihood of preterm birth, such as bacterial infection/colonization, maternal stress, and genetic predisposition. While these factors increase the frequency of preterm birth, the majority of women with these factors in isolation deliver at term. In this proposal, we will test a new hypothesis - similar to insights that have been established in cancer biology - that to manifest a preterm delivery, multiple detrimental hits acting together are required. Proving that this is the case is not possible with observational studies in humans, with many uncontrollable variables confounding causal relationships. We will exploit a non-human primate (rhesus) model system, with pregnancy characteristics more similar to humans than typical non-primate systems, to determine whether stress and infection interact to promote early labor and delivery. We propose that individual temperament, inflammation, and stress will each provide an additive hit, of which two or more will be required to end pregnancy prematurely. We will test the specific hypotheses that: (1) maternal stress and inflammation synergize to induce preterm birth; (2) the individual susceptibility to psychological stressors plays a key role in the induction of preterm birth; and (3) maternal peripheral blood or amniotic fluid hormones and inflammatory responses will differ prior to and following IL-1 administration during pregnancy depending on underlying temperament and exposure to chronic stress. To test these hypotheses, our Specific Aims will determine: (1) The interactions between maternal stress, inflammation, and the influence of individual susceptibility due to anxious temperament, in preterm birth in rhesus macaques. (2) The interactions of maternal temperament and stress on maternal immunity and hormones before and after an inflammatory challenge. (3) The effects of maternal temperament and chronic stress on amniotic fluid cytokines, prostaglandins and microbial community structure before and after an inflammatory challenge. Our transdisciplinary team will integrate expertise in the physiology of pregnancy, immunology/inflammation, primate behavior/psychology, the neurobiology of stress, biostatistics and the microbiome to more comprehensively investigate the heterogeneous pathways increasing preterm birth risk and yield important new insights into causal mechanisms and avenues for prematurity prevention.